The overall goal of this project is to investigate the contribution of amino acid side-chain dynamics to the physico-chemical mechanisms that determine the thermodynamics of recognition and association in protein/DNA interactions and the molecular basis of cooperativity of ion binding in calcium-binding proteins (CaBPs). Side chains can make significant contributions to the configurational entropy of a protein, thereby modulating the thermodynamics of protein function. A fundamental understanding of how proteins work therefore requires an intimate knowledge of the dynamic properties of the side chains. Two model systems, protein/DNA complexes and the CaBP calbindin D9k, have been selected for study that will allow key insights to be obtained regarding the role of side-chain dynamics in protein/DNA binding/recognition and cooperativity of ion binding, respectively. Despite the large number of structural and thermodynamic studies that have been reported for a variety of protein/DNA systems, critical and substantial gaps exist in our knowledge and understanding of the role played by molecular dynamics in protein/DNA interactions. A general problem in the field of molecular recognition is that structural studies reveal relatively little about the entropic component of the free energy of complex formation. Thus, it is very important to complement structural information by undertaking studies to investigate side-chain dynamics in the protein/DNA interface. Cooperative ion binding is one of the fundamental properties of calcium signaling pathways. The readout of intracellular calcium signals must be very finely tuned to effect a rapid response to the transient and subtle variations in Ca2+ concentrations that constitute the calcium signals. The great importance of cooperative binding of Ca2+ by EF-hand CaBPs has motivated efforts to determine the molecular basis for cooperativity in specific members of this protein family. Calbindin D9k, a single domain EF-hand CaBP, is one of the primary model systems for studying the cooperative binding phenomenon. The general hypotheses of the proposed research are that modulation of protein side-chain dynamics plays important roles in establishing a complementary interface between consensus/non-consensus DNA sequences and a cognate DNA-binding protein, and in promoting allosteric communication between ion- binding sites that leads to cooperative calcium binding. To test these hypotheses the following specific aims are proposed: (1) determine the side-chain dynamics and thermodynamic properties of the K50-class homeodomains from the human Pitx2 and the Drosophila Bicoid proteins, bound to a consensus duplex DNA site; (2) determine the structure, dynamics and thermodynamics of the Pitx2 and Bicoid homeodomains bound to non-consensus DNA sites; (3) investigate the molecular basis and driving forces for cooperative binding of Ca2+ by the CaBP calbindin D9k; and (4) characterize the thermodynamic role of side-chain dynamics in the single-strand DNA-binding family of Telomere End Protection (TEP) proteins.